ADENOSINE AND ADENOSINE RECEPTORS
Adenosine is a nucleoside with wide distribution in the body. Adenosine mediates a broad array of physiological responses, including central nervous system sedation, inhibition of platelet aggregation and vascular smooth muscle vasodilatation. These effects occur largely through interaction of adenosine with one of two types of adenosine receptors.
Adenosine receptors comprise a group of cell surface molecules that mediate the physiologic effects of adenosine. (For recent reviews, see Stiles, G. L., Trends in Pharmacol. Sci. 7:486-490 (1986); Ramkumar, V. et al., Prog. Drug Res. 32:195-247 (1988); Olah, M. E. et al., Annu. Rev. Physiol. 54:211-225 (1992); Stiles, G. L., J. Biol. Chem. 267:6451-6454 (1992); Jacobson, K. A. et al., J. Med. Chem. 35:407-422 (1992)). This family of receptors was originally classified as P.sub.1 or P.sub.2 purinergic receptors, depending on their preferential interactions with adenosine (P.sub.1) or ATP (P.sub.2) (Burnstock, G., In: CELL MEMBRANE RECEPTORS FOR DRUGS AND HORMONES, Straub et al., eds., Raven Press, New York, 1978, pp. 107-118). The P.sub.1 sites were further subdivided into A.sub.1 and A.sub.2 adenosine receptors based on their differential selectivity for adenosine analogues (Van Calker, D. et al., J. Neurochem. 33:999-1005 (1979); Londos, C. et al., Proc. Natl. Acad. Sci. USA 77:2552-2554 (1980)). The A.sub.1 adenosine receptor, which is inhibitory to adenylyl cyclase, exhibits the potency order (R)-PIA&gt;NECA&gt;(S)-PIA. The A.sub.2 adenosine receptor, which is stimulatory to adenylyl cyclase, has a different potency order where NECA&gt;(R)-PIA&gt;(S)-PIA. ((R)-PIA is N.sup.6 -(R)-phenylisopropyladenosine; (S)-PIA is N.sup.6 -(S)-phenylisopropyladenosine; NECA is N-ethyladenosine-5'-uronic acid) Both the A.sub.1 and A.sub.2 adenosine receptors are widely distributed in the central nervous system and peripheral tissues (Ramkumar, V. et al., supra).
Until relatively recently, no truly useful radioligand was available for characterizing the A.sub.2 adenosine receptor. Demonstration of adenosine receptors in smooth muscle was made primarily by functional assays, for example, adenosine stimulation of adenylyl cyclase activity via A.sub.2 receptors in vascular smooth muscle cells in culture (Anand-Srivastava, M. B. et al., Biochem. Biophys. Res. Comm. 108:213-219 (1982); Anand-Srivastava, M. B. et al., Life Sci. 37:857-867 (1985)). However, the concentrations of adenosine required to elevate cAMP were higher than those required for full vasorelaxation in vivo (Berne, R. M., Circ. Res. 47:807-813 (1980); Herlihy, J. T. et al., Am. J. Physiol. 230:1239-1243 (1976)). One cell line which has proved useful for study of A.sub.1 and A.sub.2 adenosine receptors (Ramkumar, V. et al., Molec. Pharmacol. 37:149-156 (1990)) is the DDT1 MF-2 line, a smooth muscle cell line derived from a steroid-induced leiomyosarcoma of the vas deferens of an adult Syrian hamster (Norris, J. S. et al., Nature 248:422-424 (1974)).
Recently, two compounds were found to be selective high affinity agonist radioligands for the A.sub.2 receptor: .sup.3 H!CGS 21680 (Jarvis, M. F. et al., J. Pharmacol. Exp. Ther. 251:888-893 (1989)) and .sup.125 I-PAPA-APEC, the full chemical name of which is (2-4-2-2-(4-aminophenyl)methylcarbonyl-amino!ethylaminocarbonyl!ethyl! phenyl!ethylamino-5'-N-ethylcarboxamidoadenosine (Barrington, W. W. et al., Proc. Natl. Acad. Sci. USA 86:6572-6576 (1989)). Use of such ligands allowed identification of the A.sub.2 binding subunit as a 45 kDa protein (on SDS-PAGE) that was clearly distinguishable from the 38 kDa A.sub.1 binding subunit. Use of the azide derivative of .sup.125 I-PAPA-APEC, a direct photoaffinity probe for the A.sub.2 receptor, allowed demonstration that the A.sub.2 binding subunit is a glycoprotein clearly different from the A.sub.1 receptor glycoprotein (Barrington, W. W. et al., Mol. Pharmacol. 38:177-183 (1990)). The A.sub.2 adenosine receptor has a single carbohydrate chain of either the complex or high mannose type.
Useful adenosine receptor agonists, in particular those with selectivity for the A.sub.2 receptor are well-known in the art. These include 2-substituted adenosine-5'-carboxamide derivatives (Hutchison, U.S. Pat. Nos. 4,968,697 and 5,034,381) and N9 cyclopentyl-substituted adenine derivative (Chen et al., U.S. Pat. No. 5,063,233). The above patents are hereby incorporated by reference in their entirety.
Adenosine and its analogues interact with neutrophils in inflammatory responses. While neutrophils are essential for limiting the spread of infection by a variety of microbes, stimulated neutrophils may damage injured tissues while en route to sites of infection or inflammation. Release of adenosine is one mechanism by which normal cells may protect themselves from activated neutrophils. Thus, one important action of adenosine and its analogues is the inhibition of generation of toxic oxygen products, including O.sub.2.sup.- and H.sub.2 O.sub.2, by interacting with A.sub.2 receptors on the neutrophil (Cronstein, B. N. et al., J. Immunol. 135:1366-1371 (1985); Roberts, P. A. et al., Biochem. J. 227:669-674 (1985); Schrier, D. J. et al., J. Immunol. 137:3284-3289); Iannone, M. A. et al., Fed. Proc. 44:580 (abstr.) (1985)). Adenosine promotes neutrophil chemotaxis via the same receptor (Cronstein, B. N. et al., supra; Rose, F. R. et al., J. Exp. Med. 167:1186-1194 (1988)). Adenosine receptor ligation regulates inflammatory responses of neutrophils triggered by immune complexes acting through the Fc.gamma. receptor (Salmon, J. E. J. Immunol. 145:2235-2240 (1990)). Specifically, activation of A.sub.2 receptors inhibited these inflammatory responses, whereas activation of A.sub.1 receptors was stimulatory. These authors noted an important role for adenosine at picomolar concentrations as a promoter, and at micromolar concentration as an inhibitor, of neutrophil responses elicited by immune complexes.
Interestingly, the immunosuppressive drug methotrexate, at low concentrations, acts as an antiinflammatory agent at least in part due to its capacity to induced adenosine release by connective tissue cells such as dermal fibroblasts or umbilical vein endothelial cells. The released adenosine interacted with the neutrophil adenosine receptors (Cronstein, B. N. et al., Proc. Natl. Acad. Sci. USA 88:2441-2445 (1991)).
The nonselective adenosine receptor agonist, 2-chloroadenosine inhibited adherence of stimulated neutrophils to endothelium, thus protecting the endothelium from inflammatory effects (Cronstein, B. N. et al., J. Clin. Invest. 78:760-770 (1986)). More recently, work from the present inventors' laboratory has demonstrated that occupancy of A.sub.2 receptors inhibits neutrophil adherence and generation of toxic metabolites, thus contributing to an anti-inflammatory function (Cronstein, B. N. et al., J. Immunol. 148:2201-2206 (1992)).
Thus, work largely from the present inventor's laboratory has led to an emerging picture of adenosine as a unique "autacoid" of inflammation that regulates the accumulation of neutrophils at sites of inflammation. While neutrophils traverse through acellular connective tissue, the low concentrations of adenosine present promote phagocytosis, migration and adherence to some, but not other, surfaces. Near foci of tissue injury, damaged cells release higher concentrations of adenosine that inhibit neutrophil adherence to cells and connective tissue substrata as well as inhibiting production of toxic oxygen metabolites by stimulated neutrophils. Thus adenosine may promote accumulation of neutrophils at sites of tissue injury or microbial invasion, a pro-inflammatory function, but may also act as a feedback regulator of inflammation at sites of tissue damage, an anti-inflammatory function (Cronstein et al., 1992, supra).
Studies by others showed that adenosine itself promoted migration of renal epithelium (Kartha, S. et al., J. Clin. Invest. 90:288-292 (1992) and vascular endothelium (Meininger, C. J. et al., Amer. J. Physiol. 255:3 pt 2:H554-562 (1988). This latter reference suggested that the effect of adenosine on chemotaxis may be mediated by a receptor (of unknown or unspecified type).